Recombinant immunotoxins are chimeric proteins in which a truncated toxin is fused to an Fv portion of an antibody. The binding activity of the Fv moiety targets the immunotoxins to antigen-positive cells, which are killed by the cytotoxic activity of the toxin moiety (Pastan I., Biochim. Biophys. Acta, 1333:C1-C6 (1997); Kreitman R. J., Curr. Opin. Immunol., 11:570-578 (1999)). For cancer therapy, a number of different recombinant immunotoxins have been produced using Fvs that bind to tumor-related antigens and differentiation antigens such as CD22 and CD25 and a 38-kDa mutant form of Pseudomonas exotoxin A (“PE38”) that lacks its cell binding domain (Chaudhary et al., Nature, 339:394-397 (1989); Brinkmann et al., Proc. Natl. Acad. Sci. U.S.A, 88:8616-8620 (1991); Kreitman et al., Blood, 83:426-434 (1994); Mansfield et al., Blood, 90:2020-2026 (1997); Kreitman et al., Int. J. Cancer, 81:148-155 (1999)). The therapeutic potency of such immunotoxins has also been improved by protein engineering and chemical modification (Reiter et al., Nat. Biotechnol., 14:1239-1245 (1996); Chowdhury et al., Nat. Biotechnol., 17:568-572 (1999); Onda et al., J. Immunol., 163:6072-6077 (1999); Tsutsumi et al., Proc. Natl. Acad. Sci. U.S.A, 97:8548-8553 (2000)). These efforts have been directed at making immunotoxins that are smaller for better tumor penetration, that are less immunogenic and less toxic to animals, that bind antigen with higher affinity, that are more stable, and that are suitable for large scale production (Kreitman R. J., Curr. Opin. Immunol., 11:570-578 (1999); Brinkmann U., In Vivo, 14:21-27 (2000)).
One of the important advances is the development of disulfide-stabilized Fv fragments (dsFv) in which one of the variable chains genetically fused with PE38 is linked with the other chain by a disulfide bond between two cysteine residues engineered in the frame work region of each chain. These immunotoxins showed greater stability in vivo and in vitro than the widely used single-chain Fv (scFv) forms (Reiter et al., supra); Brinkmann et al., Proc. Natl. Acad. Sci. USA, 90:7538-7542 (1993); Reiter et al., J. Biol. Chem., 269:18327-18331 (1994); Reiter et al., Int. J. Cancer, 67:113-123 (1996)).
Recent clinical trials indicate that targeted therapy by recombinant immunotoxins shows great promise especially for some types of hematologic malignancies. The anti-CD25 scFv immunotoxin, LMB-2, produced major clinical responses in various types of leukemia and lymphoma (Kreitman et al., J. Clin. Oncol., 18:1622-1636 (2000)), and the anti-CD22 immunotoxin, RFB4(dsFv)-PE38, gave a remarkably high rate of complete remissions in patients with Hairy cell leukemia (Kreitman et al., Clin. Cancer Res., 6:1476-1487 (2000); Kreitman et al., N. Engl. J. Med., 345:241-247 (2001).
To extend the usefulness of immunotoxin therapy, it is important to develop immunotoxins against different targets. CD30 is a member of the tumor necrosis factor receptor super family. CD30 is an excellent target because it is usually highly expressed on malignant Reed Sternberg cells of Hodgkin's lymphoma (HL) and in anaplastic large cell lymphomas (ALCL), whereas it is only expressed in a small subset of normal lymphocytes and these can be resupplied from stem cells (Koon et al., Curr. Opin. Oncol., 12:588-593 (2000)). Although its function is largely unknown, CD30 has been implicated both in cell death and proliferation (Lee et al., J. Exp. Med, 183:669-674 (1996); Wiley et al., J. Immunol., 157:3635-3639 (1996); Mir et al., Blood, 96:4307-4312 (2000)). The possibility of using CD30 as a target for immunotoxin therapy has been investigated in earlier studies using anti-CD30 monoclonal antibodies (MAbs) chemically conjugated with toxins (Engert et al., Cancer Res., 50:2929-2935 (1990); Terenzi et al., Br. J. Haematol., 92:872-879 (1996); Engert et al., Int. J. Cancer, 63:304-309 (1995); Pasqualucci et al., Blood, 85:2139-2146 (1995)).
To obtain an anti-CD30 immunotoxin with better properties, recombinant immunotoxins have been produced. Klimka et al. reported the production of a recombinant immunotoxin derived from the anti-CD30 MAb Ki-4 by fusing its scFv to truncated PE (Klimka et al., Br. J. Cancer, 80:1214-1222 (1999)). Recently, the anti-tumor activity of this immunotoxin was reported in a SCID mouse model (Barth et al., Blood, 95:3909-3914 (2000)). The isolation of a new anti-CD30 scFv using the phage display technique and the properties of immunotoxins containing the scFv was also reported in Rozemuller et al., Int. J. Cancer, 92:861-870 (2001). All these recombinant immunotoxins showed specific binding to CD30-positive lymphoma cell lines and killed target cells as assessed by inhibition of protein synthesis with a 50% inhibition concentration (IC50) of 40-50 pM. The cytotoxic activities were, however, much less than an immunotoxin that targets CD25 on these cells, which has an IC50 of 0.2 pM (Reiter et al., Clin. Cancer Res., 2:245-252 (1996)). Only a limited number of anti-CD30 Fvs have been suitable for making recombinant immunotoxins, and even with respect to these, the cytotoxic activities of the immunotoxins was only moderate.
The ability of an immunotoxin to kill a target cell is dependent on internalization. Although improving the affinity of the targeting portion of the immunotoxin is helpful, since this tends to increase the time the immunotoxin binds to the cell and therefore improves its opportunity to be internalized, affinity of the targeting portion of the immunotoxin, by itself, does not necessarily correlate with the immunotoxins' cell-killing ability. For example, the immunotoxin can be directed to a lysosome, where it is degraded, rather than to the cytsosol, where the toxin can be released. Unfortunately, the state of the art does not yet permit predicting which antibodies will make good immunotoxins. Further, CD30 undergoes proteolytic cleavage, resulting in the release of a soluble portion of the protein, known as “sCD30.” Immunotoxins which bind to sCD30 cleaved from intact CD30 are not available to be internalized into a target cell. Immunotoxins whose targeting portion binds to sCD30 must therefore be administered in larger quantities than might otherwise be desirable to compensate for loss of some of the immunotoxin by binding by free sCD30 in the extracellular fluids, such as the serum. This problem also extends to other immunoconjugates, such as a radioisotope attached to an antibody, to the extent that their cell killing or labeling abilities are reduced by binding to free sCD30 in the circulation. There remains a need in the art for immunotoxins directed against the CD30 antigen which have high cytotoxicity to target cells or which bind to intact CD30 but not to sCD30.